Protein-protein interactions are central to many biological processes and hence represent a large and important class of targets for human therapeutics (Arkin et al., Nature Reviews Drug Discovery, 2004. 3(4):301-317; Berg, Angewandte Chemie-International Edition, 2003. 42(22):2462-2481). Recent discovery of a variety of low-molecular-weight compounds that interfere with biologically relevant protein-protein complexes has fostered the identification and validation of new therapy strategies for a variety of diseases (Arkin, Current Opinion in Chemical Biology, 2005. 9(3):317-324). Nevertheless, disrupting or modulating protein-protein interactions (PPIs) with low-molecular-weight compounds is extremely difficult due to the lack of deep binding pockets on protein surfaces. The adaptive nature of binding sites on protein surfaces creates additional challenges for lead compound design. Furthermore, because PPIs occur over a large surface area, it is difficult to identify potent and specific Protein-Protein Interaction Modulators (PPIMs) by conventional high throughput screening (HTS) of small molecule libraries.
Over the past 15 years, a variety of fragment-based lead discovery approaches have been developed and successfully applied for the development of potent PPIMs. [Albert et al., Current Topics in Medicinal Chemistry, 2007, 7(16):1600-1629; Erlanson, Current Opinion in Biotechnology, 2006. 17(6):643-652; Carr et al., Drug Discovery Today, 2005. 10(14):987-99). These approaches are commonly based on the detection of fragments binding to the target protein followed by the study of their binding to the protein target at atomic level resolution using X-ray crystallography or NMR spectroscopy. The initial hits are further optimized via fragment growing, in which fragments are extended into identified binding sites step-by-step, or via fragment linking, in which fragments identified to bind to adjacent binding sites are covalently linked together. (Poulsen et al., Bioorganic & Medicinal Chemistry, 2006. 14(10):3275-3284; Schulz et al., Current Opinion in Pharmacology, 2009. 9(5):615-621; Congreve et al., Journal of Medicinal Chemistry, 2008. 51(13):3661-3680).
Even though fragment-based discovery strategies have been very successful for the development of PPIMs, they are mainly limited by two constraints. Detection and quantification of fragment binding requires specially designed methodology due to the weak binding typically observed for fragments. Furthermore, the optimization of fragments into potent and selective compounds is not straightforward and not rapidly achievable, even though structural information is available. (Schulz et al., Current opinion in pharmacology, 2009. 9(5):615-21; Murray et al., Nature Chemistry, 2009. 1(3):187-192). For example, though good quality NMR structures were available, the well-known development of Bcl-XL PPIMs by Abbott required several design iterations and the preparation and testing of more than thousand compounds in order to yield ABT-737 (Oltersdorf, et al., Nature, 2005. 435(7042):677-681; Wendt, et al., Journal of Medicinal Chemistry, 2006. 49(3):1165-1181; Hajduk, Journal of Medicinal Chemistry, 2006. 49(24): 6972-6976). Furthermore, of the very first design consisting of 21 different compounds containing the structural motifs of the initial fragments identified by NMR, most compounds bound to Bcl-XL with a dissociation constant greater than 10 mM. (Petros, et al., Journal of Medicinal Chemistry, 2006. 49(2):656-663).
Recently, fragment-based discovery strategies have been reported which involve the protein target directly to select and assemble its own inhibitory compounds from pools of reactive fragments. These approaches, also termed in situ click chemistry or kinetic TGS approaches, were conceptually described in detail in the 1980s and are still relatively unexplored compared to dynamic combinatorial chemistry (Sharpless et al., Expert Opin. Drug Discovery, 2006. 1(6):525-538; Hu et al., Chemical Society Reviews, 2010. 39(4):1316-1324; Jencks, Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences, 1981. 78(7): 4046-4050). Thus far kinetic TGS has mainly been applied to the identification of potent enzyme inhibitors, nevertheless kinetic TGS offers an attractive approach to PPIM lead discovery because it allows the protein to select and combine building blocks that fit best into its binding sites, thus assembling larger compounds (Sharpless et al., Expert Opin. Drug Discovery, 2006. 1(6):525-538; Hu et al., Chemical Society Reviews, 2010. 39(4):1316-1324). The screening method can be as simple as determining whether or not the PPIM product has been formed in a given test mixture. Additionally, if one considers a protein target to be an ensemble of interconverting conformers, it is easy to imagine this protein undergoing dynamic motion to repeatedly expose unique structural elements vulnerable to strong binding by the right inhibitor. Unfortunately, such short-lived targets of opportunity cannot be “seen” or easily discovered with present techniques.